ScipionTomo: Towards cryo-electron tomography software integration, reproducibility, and validation.

Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline. The Scipion framework, originally developed for SPA (de la Rosa-Trevín et al., 2016), has a generic python workflow engine that gives it the versatility to be extended to other fields, as demonstrated for model building (Martínez et al., 2020). In this article, we provide an extension of Scipion based on a set of tomography plugins (referred to as ScipionTomo hereafter), with a similar purpose: to allow users to be focused on the data processing and analysis instead of having to deal with multiple software installation issues and the inconvenience of switching from one to another, converting metadata files, managing possible incompatibilities, scripting (writing a simple program in a language that the computer must convert to machine language each time the program is run), etcetera. Additionally, having all the software available in an integrated platform allows comparing the results of different algorithms trying to solve the same problem. In this way, the commonalities and differences between estimated parameters shed light on which results can be more trusted than others. ScipionTomo is developed by a collaborative multidisciplinary team composed of Scipion team engineers, structural biologists, and in some cases, the developers whose software packages have been integrated. It is open to anyone in the field willing to contribute to this project. The result is a framework extension that combines the acquired knowledge of Scipion developers in close collaboration with third-party developers, and the on-demand design of functionalities requested by beta testers applying this solution to actual biological problems.

On bias, variance, overfitting, gold standard and consensus in single-particle analysis by cryo-electron microscopy.

Cryo-electron microscopy (cryoEM) has become a well established technique to elucidate the 3D structures of biological macromolecules. Projection images from thousands of macromolecules that are assumed to be structurally identical are combined into a single 3D map representing the Coulomb potential of the macromolecule under study. This article discusses possible caveats along the image-processing path and how to avoid them to obtain a reliable 3D structure. Some of these problems are very well known in the community. These may be referred to as sample-related (such as specimen denaturation at interfaces or non-uniform projection geometry leading to underrepresented projection directions). The rest are related to the algorithms used. While some have been discussed in depth in the literature, such as the use of an incorrect initial volume, others have received much less attention. However, they are fundamental in any data-analysis approach. Chiefly among them, instabilities in estimating many of the key parameters that are required for a correct 3D reconstruction that occur all along the processing workflow are referred to, which may significantly affect the reliability of the whole process. In the field, the term overfitting has been coined to refer to some particular kinds of artifacts. It is argued that overfitting is a statistical bias in key parameter-estimation steps in the 3D reconstruction process, including intrinsic algorithmic bias. It is also shown that common tools (Fourier shell correlation) and strategies (gold standard) that are normally used to detect or prevent overfitting do not fully protect against it. Alternatively, it is proposed that detecting the bias that leads to overfitting is much easier when addressed at the level of parameter estimation, rather than detecting it once the particle images have been combined into a 3D map. Comparing the results from multiple algorithms (or at least, independent executions of the same algorithm) can detect parameter bias. These multiple executions could then be averaged to give a lower variance estimate of the underlying parameters.

Cryo-EM and Single-Particle Analysis with Scipion.

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Algorithmic robustness to preferred orientations in single particle analysis by CryoEM.

The presence of preferred orientations in single particle analysis (SPA) by cryo-Electron Microscopy (cryoEM) is currently one of the hurdles preventing many structural analyses from yielding high-resolution structures. Although the existence of preferred orientations is mostly related to the grid preparation, in this technical note, we show that some image processing algorithms used for angular assignment and three-dimensional (3D) reconstruction are more robust than others to these detrimental conditions. We exemplify this argument with three different data sets in which the presence of preferred orientations hindered achieving a 3D reconstruction without artifacts or, even worse, a 3D reconstruction could never be achieved.

Effectiveness of ustekinumab in patients with psoriatic arthritis in a real-world, multicenter study.

OBJECTIVE: To assess the effectiveness and survival of ustekinumab (UST) among patients with psoriatic arthritis (PsA) treated under routine clinical care. METHODS: Multicenter study. Epidemiological and clinical data was collected through electronic medical records of all patients with PsA who started UST in 15 hospitals of Spain. RESULTS: Two hundred and one patients were included, 130 (64.7%) with 45 mg and 71 (35.3%) with 90 mg. One hundred and thirty one patients (65.2%) had previously received another biological therapy. The median baseline DAS 28 ESR was 3.99, and Psoriasis Area and Severity Index (PASI) was 3. Overall, there was a significant decrease in DAS66/68 CRP, swollen joint count (SJC), tender joint count (TJC), and PASI in the first month of treatment, with earlier improvement in skin (PASI) than joints outcomes. Survival was numerically lower in patients with UST 45 mg (58.1%) than 90 mg (76.1%), although significant differences were not found (p = 0.147). When comparing naïve and < 1 TNF blocker versus > 2 TNF blocker-experienced patients, a significantly earlier response was seen in the former group regarding SJC (p = 0.029) at 1 month. Fifty-one patients (25.3%) stopped UST due to joint inefficacy and 4 patients due to adverse events (1.9%). Drug survival was significantly better in patients with fewer lines of previous biological agents (p = 0.003 for < 1 TNF blocker versus > 2 TNF blocker users). CONCLUSIONS: UST was effective in PsA patients in a routine clinical care setting. Patients with UST 90 mg and fewer lines of previous biologics achieved better and faster responses. Key Points • Largest cohort of patients with PsA in treatment with UST with specific rheumatological indication. • First cohort of patients with PsA comparing effectiveness of UST according to 45/90 mg dose.

Monitoring spatiotemporal changes in chaperone-mediated autophagy in vivo.

Autophagy malfunctioning occurs in multiple human disorders, making attractive the idea of chemically modulating it with therapeutic purposes. However, for many types of autophagy, a clear understanding of tissue-specific differences in their activity and regulation is missing because of lack of methods to monitor these processes in vivo. Chaperone-mediated autophagy (CMA) is a selective type of autophagy that until now has only been studied in vitro and not in the tissue context at single cell resolution. Here, we develop a transgenic reporter mouse that allows dynamic measurement of CMA activity in vivo using image-based procedures. We identify previously unknown spatial and temporal differences in CMA activity in multiple organs and in response to stress. We illustrate the versatility of this model for monitoring CMA in live animals, organotypic cultures and cell cultures from these mice, and provide practical examples of multiorgan response to drugs that modulate CMA.

Integration of Cryo-EM Model Building Software in Scipion.

Advances in cryo-electron microscopy (cryo-EM) have made it possible to obtain structures of large biological macromolecules at near-atomic resolution. This "resolution revolution" has encouraged the use and development of modeling tools able to produce high-quality atomic models from cryo-EM density maps. Unfortunately, many practical problems appear when combining different packages in the same processing workflow, which make difficult the use of these tools by non-experts and, therefore, reduce their utility. We present here a major extension of the image processing framework Scipion that provides inter-package integration in the model building area and full tracking of the complete workflow, from image processing to structure validation.

Analysis of Chaperone-Mediated Autophagy.

Chaperone-mediated autophagy (CMA) is a selective type of autophagy whereby a specific subset of intracellular proteins is targeted to the lysosome for degradation. These proteins are identified by a chaperone that targets them to lysosomes. There, they are translocated into the organelle lumen through a lysosomal membrane receptor/translocation complex. CMA plays an important role in maintaining cellular proteostasis by eliminating damaged and altered proteins. CMA also participates in the control of the cellular energetic balance through recycling of amino acids resulting from lysosomal proteolysis of the substrate proteins. Lastly, due to the intrinsic protein selectivity of CMA, this type of autophagy exerts regulatory functions by mediating timely degradation of key cellular proteins that participate in processes such as lipid and glucose metabolism, cell cycle, DNA repair, and cellular reprogramming, among others. Dysfunctional CMA occurs with age and has now been described in a growing list of human pathologies such as metabolic disorders, neurodegeneration, cancer, immunodeficiency, and diabetes. In this chapter, we describe current methodologies to quantitatively analyze CMA activity in different experimental models.

BAHNG score: predictive model for detection of subjects with the oropharynx colonized by uncommon microorganisms.

OBJECTIVE: Pneumonia is most frequently produced by the microaspiration of flora that colonizes the oropharynx. Etiological diagnosis of pneumonia is infrequent in clinical practise and empirical treatment should be prescribed. The aims of the present study were to determine the factors associated with oropharynx colonization by uncommon microorganisms (UM) and to develop a predictive model. METHODS: A cross-sectional study that included all pa-tients living in one long-term care facilities was developed. Demographic, comorbidities, basal functional status and clinical data were collected. To determinate the oropharyngeal colonization, a single sample of pharynx was obtained for each subject using a cotton swab. RESULTS: A total of 221 subjects were included, mean age 86.27 (SD 8.05) years and 157 (71%) were female. In 32 (14.5%) subjects UM flora was isolated, Gram-negative bacilli in 16 (7.2%) residents, and Staphylococcus aureus in 16 (7.2%). The predictive model included the presence of hypertension, neuromuscular disease, Barthel <90 and use of PEG. The BAHNG score (BArthel, Hypertension, Neuromuscular, Gastrostomy), showed an area under the curve of 0.731 (CI 95% 0.643-0.820; p<0.001). We have classified patients according to this score in low (0-2 points), intermediate (3-5 points) and high risk (≥ 6). The probability of UM colonization in the oropharyngeal based on this classification is 4.1%, 15.8% and 57.1% for low, intermediate and high risk, respectively. CONCLUSIONS: The BAHNG score could help in the identifications of elderly patients with high risk of colonization by UM. In case of pneumonia the evaluation of the subject through this score could help in the initial decisions concerning antibiotic treatment.

The relationship between phenolic compounds from diet and microbiota: impact on human health.

The human intestinal tract is home to a complex microbial community called microbiota. This gut microbiota, whilst playing essential roles in the maintenance of the health of the host, is exposed to the impact of external factors such as the use of medication or dietary patterns. Alterations in the composition and/or function of the microbiota have been described in several disease states, underlining the role of the gut microbiota in keeping the health status. Among the different dietary compounds, polyphenols constitute a very interesting group as some of them have been found to possess important biological activities, including antioxidant, anticarcinogenic or antimicrobial activities. The term polyphenol comprises thousands of molecules presenting a phenol ring and are widely distributed in plant foods. The bioactivity of these compounds is highly dependent on their intestinal absorption and often they are ingested as non-absorbable precursors that are transformed into bioactive forms by specific microorganisms in the intestine. Some of these microorganisms have been identified and the enzymatic steps involved have been elucidated. However, little is known about the impact of these ingested polyphenols upon the human gut microbiota. The heterogeneity of the polyphenol compounds and their food sources, as well as their coexistence with other bioactive compounds within a normal diet, together with the complexity of the human gut microbiota make difficult the understanding of the interactions between dietary polyphenols and gut microbes. This is, however, an important area of research which promises to expand our knowledge on the food functionality area through understanding the microbiota-food component interaction.

Autophagy and regulation of cilia function and assembly.

Motile and primary cilia (PC) are microtubule-based structures located at the cell surface of many cell types. Cilia govern cellular functions ranging from motility to integration of mechanical and chemical signaling from the environment. Recent studies highlight the interplay between cilia and autophagy, a conserved cellular process responsible for intracellular degradation. Signaling from the PC recruits the autophagic machinery to trigger autophagosome formation. Conversely, autophagy regulates ciliogenesis by controlling the levels of ciliary proteins. The cross talk between autophagy and ciliated structures is a novel aspect of cell biology with major implications in development, physiology and human pathologies related to defects in cilium function.

Dietary intake of polyphenols and major food sources in an institutionalised elderly population.

BACKGROUND: Polyphenols are bioactive compounds widely found in fruit, vegetables and beverages of plant origin. Epidemiological studies have suggested an association between polyphenol intake and health; antioxidant, anti-inflammatory, anti-carcinogenic and other bioactivities may contribute to these beneficially protective effects. To date, most epidemiological studies describing polyphenol intake have been limited by the information available in nutrient databases. The present study aimed to determine the total and individual polyphenol intake among institutionalised elderly people living in Asturias (North of Spain) and to identify the major dietary sources of polyphenol classes and subclasses. METHODS: The study sample comprised 304 subjects with a mean age of 73.2 years for men and 76.8 years for women. Dietary intake was assessed by means of a food frequency questionnaire. Phenol content was estimated from the Phenol-Explorer database, as developed at the French National Institute for Agricultural Research. The contribution of each food to the total and subgroup intake of polyphenols was calculated as a percentage. RESULTS: Except for flavonones, total polyphenol intake, groups and subgroups, was higher in men than women. The main polyphenol groups contributing to total polyphenol intake were flavonoids (62%) and phenolic acids (35.5%). We identified red wine, coffee, apples, oranges and green beans as the major food sources providing total polyphenol intake. Flavonoid and lignan intake was lower for those aged >80 years. Smoking habit, red wine consumption, physical activity and a Mediterranean diet score were associated with a greater polyphenol intake. CONCLUSIONS: The present study provides information on polyphenol intake in an elderly Mediterranean population with a level of detail that has not been achieved previously. The identification of age and lifestyle factors associated with the intake of polyphenols may be useful in future studies regarding polyphenols.

Autophagic pathways and metabolic stress.

Autophagy is an essential intracellular process that mediates degradation of intracellular proteins and organelles in lysosomes. Autophagy was initially identified for its role as alternative source of energy when nutrients are scarce but, in recent years, a previously unknown role for this degradative pathway in the cellular response to stress has gained considerable attention. In this review, we focus on the novel findings linking autophagic function with metabolic stress resulting either from proteins or lipids. Proper autophagic activity is required in the cellular defense against proteotoxicity arising in the cytosol and also in the endoplasmic reticulum, where a vast amount of proteins are synthesized and folded. In addition, autophagy contributes to mobilization of intracellular lipid stores and may be central to lipid metabolism in certain cellular conditions. In this review, we focus on the interrelation between autophagy and different types of metabolic stress, specifically the stress resulting from the presence of misbehaving proteins within the cytosol or in the endoplasmic reticulum and the stress following a lipogenic challenge. We also comment on the consequences that chronic exposure to these metabolic stressors could have on autophagic function and on how this effect may underlie the basis of some common metabolic disorders.

An in-tube SPME device for the selective determination of chlorophyll a in aquatic systems.

We report a new device for the estimation of the content of chlorophyll a pigment in water samples as an indicator of water quality. The extraction of the pigment from water was also optimized. 10 mL of water was filtered through a nylon filter (45 microm pore size and 13 mm of diameter), after the chlorophylls were dissolved by immersing the filter in 1 mL of a low non-hazardous solvent as ethanol. An in-valve in-tube SPME device coupled to capillary liquid chromatography with diode array detection was designed. A capillary column of 70 cm in length (0.32 mm i.d. coated with 5% diphenyl-95% polydimethylsiloxane, 3 microm coating thickness) was used as the loop of the injection valve for preconcentration and a Zorbax SB C(18) (SiO(2)-based) 150 mm x 0.5 mm i.d., 5 microm column (Agilent) was used as analytical column. The achieved detection limit was 0.05 microg L(-1) and the working range of concentrations was 0.1-1 microg L(-1). % RSD values between 2 and 11 were obtained. Chlorophyll a in several water matrices was determined with good results in presence of other pigments such as chlorophyll b, pheophytin a and pheophytin b.

Chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is the only type of autophagy in mammalian cells able to selectively degrade cytosolic proteins in lysosomes. CMA is maximally activated in response to stressors such as prolonged starvation, exposure to toxic compounds, or oxidative stress. We have found that CMA activity decreases in aging and in some age-related disorders such as Parkinson's disease. Impaired CMA under these conditions may be responsible for the accumulation of damaged proteins inside cells and for their higher vulnerability to stressors. In contrast to other forms of autophagy, where substrates are engulfed or sequestered along with other cytosolic components, CMA substrates are translocated one-by-one across the lysosomal membrane. Changes in the levels/activity of the lysosomal components required for substrate translocation can be used to stimulate CMA activity. However, the most unequivocal method to measure CMA is by directly tracking the translocation of substrate proteins into isolated lysosomes.

Cuantificación de hongos micorrícicos en muestras de suelo en plantaciones de Tabebuia rosea y Cordia alliodora / Quantification of mycorrhizal fungi in ground samples in plantations of Tabebuia rosea and Cordia alliodora

Después de que han sido intervenidos y sobreexplotados por el hombre; los suelos tropicales presentan grandes limitantes para la producción de árboles. Una estrategia para aumentar los niveles de producción se basa en la fertilización acorde con los requerimientos de los árboles. Como la micorrización es una alternativa biológica de bajo costo, que puede contribuir al éxito de la repoblación forestal, se quiso establecer cómo se podría favorecer la sobrevivencia y el crecimiento de especies arbóreas en viveros por medio de la cuantificación de hongos micorricicos utilizando muestras de suelo en las especies Tabebuia rosea y Cordia alliodora en diferentes localidades de Costa Rica, Centro América. Se analizaron muestras en diez sitios diferentes. La cuantificación de esporas se realizó mediante la técnica de tamizado y el porcentaje de colonización de raíces se estimó mediante la observación microscópica de raíces según metodología de Sieverding. Los resultados obtenidos en esta investigación permitieron concluir que, de la variedad de los géneros de Micorrizas Vesiculo Arbusculares presentes en la muestra de suelo, los que demostraron mejor comportamiento con Tabebuia rosea y Cordia. alliodora fueron Glomus sp y Gigaspora sp. Además para lograr un mayor crecimiento y desarrollo de los árboles, es necesario favorecer la población micorrizal en las plantas en sus primeros días de nacidas.

[Good practice guidelines for the treatment of autistic spectrum disorders]. / Guia de buena practica para el tratamiento de los trastornos del espectro autista.

INTRODUCTION: Due to the inexistence of an aetiology-based intervention for autistic spectrum disorders (ASD) families and professionals are exposed to diverse and sometimes conflictive recommendations when they have to decide the most adequate alternative for treatment. AIM: To elaborate treatment guidelines agreed by consensus at the ASD Study Group of the (National) Institute of Health Carlos III. DEVELOPMENT: Information about treatment of ASD was searched and gathered through available evidence based medical (EBM) databases. The data generated was complemented with practice parameters published elsewhere, reports from prestigious international institutions, focus oriented searches in PubMed and, finally, the opinion and experience of a multidisciplinary Study Group with extensive experience in treating ASD in Spain. Most popular treatment methods were reviewed as well as the common elements to be considered in successful support programs. CONCLUSION: No simple treatment algorithm can be produced at this time, and the level of available evidence based recommendations are in the weaker degrees of EBM classifications. Nevertheless, there is widespread agreement to stress that education, with special incidence in the development of communication and social competence, with the addition of community support are the main means of treatment. They can be complemented, depending on individual needs, with medication, behavioural approaches and cognitive-behavioural therapy for associated psychological problems in persons with higher cognitive level. Support to families and community empowerment are essential elements for the quality of life of persons with ASD.

Autophagy as a cell-repair mechanism: activation of chaperone-mediated autophagy during oxidative stress.

Proper removal of oxidized proteins is an important determinant of success when evaluating the ability of cells to handle oxidative stress. The ubiquitin/proteasome system has been considered the main responsible mechanism for the removal of oxidized proteins, as it can discriminate between normal and altered proteins, and selectively target the latter ones for degradation. A possible role for lysosomes, the other major intracellular proteolytic system, in the removal of oxidized proteins has been often refused, mostly on the basis of the lack of selectivity of this system. Although most of the degradation of intracellular components in lysosomes (autophagy) takes place through "in bulk" sequestration of complete cytosolic regions, selective targeting of proteins to lysosomes for their degradation is also possible via what is known as chaperone-mediated autophagy (CMA). In this work, we review recent evidence supporting the participation of CMA in the clearance of oxidized proteins in the forefront of the cellular response to oxidative stress. The consequences of an impairment in CMA activity, observed during aging and in some age-related disorders, are also discussed.

[Best practice guidelines for research in autistic spectrum disorders]. / Guía de buena práctica para la investigación de los trastornos del espectro autista.

INTRODUCTION: Achieving a better knowledge of autism and other pervasive developmental disorders known as autistic spectrum disorders (ASD), poses a major scientific challenge. These disorders are some of the earliest and most severe psychopathological disorders in infancy; they include an heterogeneous group of conditions; its prevalence rate seems to be continually increasing and they generate a significant social impact. AIMS AND DEVELOPMENT: Nowadays, there is a current international agreement on the general requirements to be fulfilled by research projects and the priority areas to be considered when developing ASD high quality research. In Spain, although there are some established research groups with broad experience and expertise in these disorders, public funding opportunities and research development are still scarce. For this reason, the Study Group of the Instituto de Salud Carlos III has generated by consensus some Good Practice Guidelines for Research in ASD. CONCLUSIONS: After comparing priorities and recommendations from international reference documents with the results obtained after having carried out an exhaustive bibliographic revision of articles published in autism in the last 30 years by Spanish authors, methodological and ethical recommendations are established. Finally, structural deficiencies to be corrected and emerging research initiatives to be supported are identified.

[Best practice guidelines for the diagnosis of autistic spectrum disorders]. / Guía de buena práctica para el diagnóstico de los trastornos del espectro autista.

INTRODUCTION: The autism spectrum disorder (ASD) diagnostic process requires expertise both in the knowledge of autism as in teamwork strategies with different professionals, often working in different clinic services, and with parents. AIM: To recommend a consensus diagnostic procedure for ASD, that has been designed by the Study Group of the Instituto de Salud Carlos III. DEVELOPMENT: The reports emphasize the need to obtain a complete clinical history, covering personal, family and psychosocial antecedents; detailing the basic areas affected in ASD--social interaction, communication and restricted patterns of behaviour, activities and interests. Diagnostic tests to be used as a routine in all cases are described and analysed--including both psychoeducational and biomedical tests. Also, tests indicated in cases with suspected identifiable physical disorders are covered, as well as those medical tests to be used for research purposes only. CONCLUSION: The diagnostic procedure requires the implementation of a coordinated interdisciplinary assessment strategy, that needs to ensure the participation of professionals from very different fields in active collaboration with the family. Their role culminates in the preparation and delivery of a personalized report. Every diagnostic procedure needs to be accompanied by an action plan that includes immediate support to the person with ASD, as well as information to the family on resources and community initiatives in their living area.